Peptides of a melanoma antigen and their use in diagnostic, prophylactic, and therapeutic methods

ABSTRACT

Immunogenic peptides of a melanoma antigen recognized by T cells, designated gp100, bioassays using the peptides to diagnose, assess or prognose a mammal afflicted with cancer, more specifically melanoma or metastatic melanoma, and use of the proteins and peptides as immunogens to inhibit, prevent or treat melanoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 10/486,989, filed May 14, 2004, now U.S. Pat. No. 7,419,957, whichis a U.S. National Phase of International Patent Application No.PCT/US02/26957, filed Aug. 22, 2002, which claims the benefit of U.S.Provisional Patent Application No. 60/314,183, filed Aug. 22, 2001.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 6,220 Byte ASCII (Text) file named“703492ST25.TXT,” created on Aug. 20, 2008.

FIELD OF THE INVENTION

This invention is in the field of the treatment of human cancers. Morespecifically, this invention relates to genes encoding melanoma antigensrecognized by T cells and their corresponding proteins and peptides andto diagnostic, prophylactic, and therapeutic applications which employthese genes, proteins, and peptides.

BACKGROUND OF THE INVENTION

Melanomas are aggressive, frequently metastatic tumors derived fromeither melanocytes or melanocyte related nevus cells (“Cellular andMolecular Immunology” (1991) (eds) Abbas A. K., Lechtman, A. H., Pober,J. S.; W.B. Saunders Company, Philadelphia: pages 340-341). Melanomasmake up approximately three percent of all skin cancers and theworldwide increase in melanoma is unsurpassed by any other neoplasm,with the exception of lung cancer in women (“Cellular and MolecularImmunology” (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J. S.;W.B. Saunders Company Philadelphia pages: 340-342; Kirkwood andAgarwala, (1993) Principles and Practice of Oncology 7:1-16). Even whenmelanoma is apparently localized to the skin, up to 30% of the patientswill develop systemic metastasis and the majority will die (Kirkwood andAgarwala, (1993) Principles and Practice of Oncology 7:1-16). Classicmodalities of treating melanoma include surgery, radiation andchemotherapy. In the past decade immunotherapy and gene therapy haveemerged as new and promising methods for treating melanoma.

T cells play an important role in tumor regression in most murine tumormodels. Tumor infiltrating lymphocytes (TIL) that recognize uniquecancer antigens can be isolated from many murine tumors. The adoptivetransfer of these TIL plus interleukin-2 can mediate the regression ofestablished lung and liver metastases (Rosenberg, S. A., et al., (1986)Science 233:1318-1321). In addition, the secretion of IFN-γ by injectedTIL significantly correlates with in vivo regression of murine tumors,suggesting activation of T cells by the tumor antigens (Barth, R. J., etal., (1991) J. Exp. Med. 173:647-658). The known ability of tumor TIL tomediate the regression of metastatic cancer in 35 to 40% of melanomapatients when adoptively transferred into patients with metastaticmelanoma attests to the clinical importance of the antigens recognized(Rosenberg, S. A., et al., (1988) N. Engl. J. Med. 319:1676-1680;Rosenberg, S. A., (1992) J. Clin. Oncol. 10:180-199).

T cell receptors on CD8⁺ T cells recognize a complex consisting of anantigenic peptide (9-10 amino acids for HLA-A2), β-2 microglobulin andclass I major histocompatibility complex (MHC) heavy chain (HLA-A, B, C,in humans). Peptides generated by digestion of endogenously synthesizedproteins are transported into the endoplastic reticulum, bound to classI MHC heavy chain and β2 microglobulin, and finally expressed in thecell surface in the groove of the class I MHC molecule. Thus, T cellscan detect molecules that originate from proteins inside cells, incontrast to antibodies that detect intact molecules expressed on thecell surface. Therefore, antigens recognized by T cells may be moreuseful than antigens recognized by antibodies.

Although emphasis is on CD8⁺ T cell responses, there is emerging supportthat CD4⁺ T cells may play an important role in anti-tumor immunity. Asreviewed by Pardoll and Topalian (Curr. Opin. Immunol. 10:588, 1998),CD4+ T cells have been demonstrated in murine studies to exert helperactivity through the induction of CD8+ T cells and B cells and furtherhave both direct and indirect effects on tumor cells, including thosedeficient in MHC class II. In humans, CD4+ T cells play a critical rolein the initiation of several autoimmune diseases (Parry et al., Curr.Opin. Immunol. 10:663, 1998) and in pathogenic resistance (Mata andPaterson, J. Immunol. 163:1449, 1999; Zajac et al., J. Exp. Med.188:2205, 1998). CD4+ T cells activated dendritic cells primarilythrough the interaction of CD40 and its ligand. There is growing supportthat the combination of MHC class I and class II epitopes derived fromthe same tumor antigen can enhance antitumor effector function andlong-term immunity (Surman et al., J. Immunol. 164:562, 2000; Ossendorpet al., J. Exp. Med. 187:693, 1998; Matloubian et al., J. Virol.68:8056, 1994).

Strong evidence that an immune response to cancer exists in humans isprovided by the existence of lymphocytes within melanoma deposits. Theselymphocytes, when isolated, are capable of recognizing specific tumorantigens on autologous and allogeneic melanomas in an MHC-restrictedfashion (Itoh, K. et al. (1986), Cancer Res. 46: 3011-3017; Muul, L. M.,et al. (1987), J. Immunol. 138:989-995); Topalian, S. L., et al., (1989)J. Immunol. 142: 3714-3725; Darrow, T. L., et al., (1989) J. Immunol.142: 3329-3335; Hom, S. S., et al., (1991) J. Immunother. 10:153-164;Kawakami, Y., et al., (1992) J. Immunol. 148: 638-643; Hom, S. S., etal., (1993) J. Immunother. 13:18-30; and O'Neil, B. H., et al., (1993)J. Immunol. 151: 1410-1418). TIL from patients with metastatic melanomarecognize shared antigens including melanocyte-melanoma lineage specifictissue antigens in vitro (Kawakami, Y., et al., (1993) J. Immunother.14: 88-93; Anichini, A. et al., (1993) et al., J. Exp. Med. 177:989-998). Anti-melanoma T cells appear to be enriched in TIL, probablyas a consequence of clonal expansion and accumulation at the tumor sitein vivo (Sensi, M., et al., (1993) J. Exp. Med. 178:1231-1246). The factthat many melanoma patients mount cellular and humoral responses againstthese tumors and that melanomas express both MHC antigens and tumorassociated antigens (TAA) suggests that identification andcharacterization of additional melanoma antigens will be important forimmunotherapy, of patients with melanoma.

The melanocyte differentiation antigen, gp100, is expressed in more than75% of human melanomas (Cormier et al., Int. J. Cancer 75:517, 1998).Although the gp100 antigen is predominantly expressed intracellularly,it is a suitable immunogenic antigen. The intracellular proteins havebeen demonstrated to be processed and presented as peptides in thecontext of MHC molecules to immune system cells. In particular, TILderived from tumors of melanoma patients have been identified and reactwith the gp100 antigen. Given that vaccination with a modified gp100CD8+ T cell epitope combined with IL-2 reportedly resulted in a 42%response rate in patients with metastatic melanoma (Rosenberg et al.,Nat. Med. 4:321, 1998; Parkhurst et al., J. Immunol. 157:2539, 1996),only a few patients responded clinically to this particular vaccineregimen, and additionally, only transient responses were observed. Thus,in order to increase the immunogenicity and therapeutic efficacy ofvaccines comprising gp100 CD8+ T cell epitopes, antigen-specific CD4+ Tcells can be combined. Therefore, the gp100 MHC class I and class IIepitopes can be useful for cellular responses against melanoma, and canalso play a significant role in therapy and diagnosis of melanomapatients.

Peripheral blood lymphocytes have been used to identify severalpotential melanoma tumor antigens. For example, Van Der Bruggen et al.(Science 254: 1643-1647, 1991) has characterized a gene coding for amelanoma antigen, designated MAGE-1, using T cell clones establishedfrom the peripheral blood of patients who were repetitively immunized invivo with mutagenized tumor cells. Cytotoxic T cells derived from theperipheral blood lymphocytes of patients with melanoma were used toidentify a potential antigenic peptide encoding MAGE-1 (Traversari, C.,et al. (1992) J. Exp. Med. 176:1453-1457). Brichard et al. ((1993) J.Exp. Med. 178:489-495) has also characterized a gene encoding a melanomaantigen designated tyrosinase using peripheral blood lymphocytes frompatients who were sensitized by repetitive in vitro stimulation withtumor. Further support for the therapeutic potential of melanomaantigens is provided by Brown et al. (U.S. Pat. No. 5,262,177). Brown etal. (U.S. Pat. No. 5,262,177) relates to a recombinant vacciniavirus-based melanoma vaccine where the melanoma antigen p97 is reportedto show a protective effect from tumor cell challenge in a murine model.Characterization of additional melanoma antigens can be important forthe development of new strategies for cancer immunotherapy, inparticular for melanoma.

SUMMARY OF THE INVENTION

This invention relates, in general, to peptides or variations ofpeptides derived from a melanoma-associated antigen known as gp100. Inaddition, the invention relates to methods of using the gp100 peptidesor derivatives thereof for treating and preventing the progression ofmelanoma-associated diseases. Further, the gp100 peptides and variantsthereof can be used as an immunogen for the treatment of patients inneed thereof.

The present invention provides immunogenic peptides derived from a gp100melanoma antigen protein sequence. For example, new peptides areexemplified by SEQ ID NOs:2-21.

In addition, the present invention provides compositions or immunogenscomprising all or part of the gp100 protein or peptides capable ofeliciting an immune response in a mammal to melanoma antigens.

Further provided are diagnostic methods for human disease involvinggp100 peptides or variants thereof, in particular for melanomas andmetastatic melanomas.

Still further provided are therapeutic methods for the treatment ofmelanoma using all or part of the gp100 peptides, variants, vaccines orimmunogens thereof.

Yet still further provided are methods of inhibiting or preventing therecurrence of melanoma in a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the generation of HLA-DR-specific T cells usinggp100-transduced dendritic cells (DC). Gp100-transduced DCs were used tostimulate autologous T cells in the presence of CD40L andlipopolysaccharide (LPS). After stimulating two times, the cultured Tcells were cloned by limiting dilution and reactivity was assessed byrecognition of autologous melanoma cells expressing gp100. (A)Reactivity of some representative clones is shown. (B) Reactivity ofclones 6 and 8 against autologous (Auto) or HLA-DR un-matched EBV-Bcells after pulsing with control protein (NY-ESO-1) or gp100 purifiedprotein is shown. Antibodies blocking MHC class I (X-MHC cl I), MHCclass II (X-MHC cl II) or HLA-DR (X-HLA-DR) recognition were added tothe assay. Results are expressed in pg/ml of IFN-γ released following a24 h co-culture. Legend: neg: negative.

FIG. 2 shows specific recognition by a T cell clone of HLA-DR matched Bcells pulsed with purified gp100. T cells were co-cultured with EBV-Bcells prepared from donors sharing different HLA-DR alleles after a 16 hpulsing with purified proteins (NY-ESO-1 or gp100). Results areexpressed in pg/ml of IFN-γ released following a 24 h co-culture.

FIGS. 3A and 3B show the identification of an HLA-DRβ1*0701gp100-epitope using an overlapping peptide library. (A) T cell clones 7and 8 were co-cultured with autologous EBV-B cells pulsed with NY-ESO-1or gp100 proteins (10 μg/ml) or 68 overlapping peptides derived fromgp100 (all at 100 μM). (B) A gp100₁₇₀₋₁₉₀-specific T cell clone wasco-cultured with autologous EBV-B cells after pulsing with gp100 proteinor the gp100₁₇₀₋₁₉₀ peptide at different concentrations. Results areexpressed in pg/ml of IFN-γ release following a 24 h co-culture.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of a more complete understanding of the invention, thefollowing definitions are described herein. Nucleic acid sequencesinclude, but are not limited to, DNA, RNA or cDNA, preferably gp100nucleic acids. Gp100 messenger RNA (mRNA) refers to one or more RNAtranscripts which are a product of the gp100 gene.

The term “melanoma” includes, but is not limited to, melanomas,metastatic melanomas, melanomas derived from either melanocytes ormelanocyte-related nevus cells, melanocarcinomas, melanoepitheliomas,melanosarcomas, melanoma in situ, superficial spreading melanoma,nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma,invasive melanoma or familial atypical mole and melanoma (FAM-M)syndrome. Such melanomas in mammals may be caused by, chromosomalabnormalities, degenerative growth and developmental disorders,mitogenic agents, ultraviolet radiation (UV), viral infections,inappropriate tissue expression of a gene, alterations in expression ofa gene, and presentation on a cell, or carcinogenic agents. Theaforementioned melanomas can be diagnosed, assessed or treated bymethods described in the present application.

“Atypical mole” means a mole with features that are abnormal and may beprecancerous.

“Major Histocompatibility Complex” (MHC) is a generic designation meantto encompass the histo-compatibility antigen systems described indifferent species, including the human leukocyte antigens (HLA).Examples of MHC Class I genes that may be used include, but are notlimited to, HLA-A, HLA-B and HLA-C genes. Examples of preferred MHCspecificities or restriction types include, but are not limited toHLA-A1, HLA-A2, such as the HLA-A2.1 subtype, or HLA-A24 (Zemmour, J. etal. (1992) Tissue Antigens 40:221-228). Further, MHC Class II genes thatare preferred include, but are not limited to, HLA-DR, HLA-DQ, andHLA-DP genes. Examples of more preferred MHC restriction types include,but are not limited to, HLA-DRβ1*0401, HLA-DRβ4-01, HLA-DRβ5-02,HLA-DRβ1-1601, and most preferably, HLA-DRβ1*0701, where 16-28% of theCaucasian and Hispanic population is HLA-DRβ1*0701.

The present invention relates to immunogenic peptides, variants,derivatives, or analogs thereof, from the gp100 protein, where thepeptide has substantially the same function as the gp100 antigen orprotein. Also encompassed by the invention are recombinant proteinsencoded by all or part of a gp100 nucleic acid sequence. One skilled inthe art would be knowledgeable of generating the gp100 proteins usingmethods commonly known. Such proteins or polypeptides include, but arenot limited to, a fragment of the gp100 protein, or a substitution,addition, or deletion mutant of a gp100 protein. This invention alsoencompasses proteins or peptides that are substantially homologous tothe gp100 melanoma antigen. “Substantially homologous” is defined hereinas about 50-100% homology, preferably about 70-100% homology, and mostpreferably about 90-100% homology between the gp100 and any anotheramino acid sequence or protein or peptide. Proteins having greater than98% homology and/or identity of sequence to gp100 (GenBank Accession No.M32295) are considered gp100 proteins and/or antigens.

An “immunogenic peptide” is defined herein as a peptide derived from thegp100 protein sequence or a gp100 protein sequence capable of causing acellular or humoral immune response in a mammal. Further, theHLA-DRβ*0701-restricted gp100 immunogenic peptides of the invention havean amino acid sequence comprising 40 amino acids or fewer in length,preferably about 10 to 30 amino acids in length, and most preferablyabout 17-21 amino acids in length.

A preferred embodiment of the invention provides immunogenic gp100peptides of at least about 40 amino acids or fewer be used, preferablyabout 40 amino acids or fewer in length, more preferably about 10-30amino acids in length, and most preferably about 17-21 amino acids inlength. A further embodiment of the invention provides an immunogenicgp100 peptide having a contiguous amino acid sequence of about 40 aminoacids or less, comprising MLGTHTMEVTV (SEQ ID NO:1), TTEWVETTARELPIPE(SEQ ID NO:21), or a variant of either of the foregoing, said peptide isHLA-DRβ1*0701 restricted and induces an immune response. Some preferredexamples of gp100 peptides of the invention comprise any of thefollowing sequences: TGRAMLGTHTMEVTVYH (SEQ ID NO:2),LSIGTGRAMLGTHTMEVTVYH (SEQ ID NO:3), IGTGRAMLGTHTMEVTVYHRR (SEQ IDNO:4), TGRAMLGTHTMEVTVYHRRGS (SEQ ID NO:5), TGRAMLGTHTMEVTVYHR (SEQ IDNO:6), TGRAMLGTHTMEVTVYHRR (SEQ ID NO:7), TGRAMLGTHTMEVTVYHRRG (SEQ IDNO:8), GTGRAMLGTHTMEVTVYHRRG (SEQ ID NO:9), GTGRAMLGTHTMEVTVYH (SEQ IDNO:10), IGTGRAMLGTHTMEVTVYH (SEQ ID NO:11), SIGTGRAMLGTHTMEVTVYH (SEQ IDNO:12), SIGTGRAMLGTHTMEVTVYHR (SEQ ID NO:13), SGLSIGTGRAMLGTHTMEVTV (SEQID NO:14), RAMLGTHTMEVTVYHRRGSRS (SEQ ID NO:15), MLGTHTMEVTVYHRRGSRSYV(SEQ ID NO:16), TGRAFLGTHTMEVTVYHRRGS (SEQ ID NO:17),TGRALLGTHTMEVTVYHRRGS (SEQ ID NO:18), TGRAYLGTHTMEVTVYHRRGS (SEQ IDNO:19), PVSGLSIGTGRAMLGTHTMEV (SEQ ID NO:20), TTEWVETTARELPIPE (SEQ IDNO:21) and a variant thereof, wherein the peptide is capable of inducingan immune response in a mammal, in particular a human.

The term “variant” as defined herein, includes any peptide whichdisplays the functional aspects of the immunogenic gp100 peptide. Anypolypeptide having an amino acid residue sequence substantiallyidentical to the gp100 sequence in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the functional aspects of the gp100 antigen is considered to bea “variant.” Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue, such as isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another, such as between arginine and lysine,between glutamine and asparagine, or between glycine and serine, thesubstitution of one basic residue, such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those proteins or peptideswhich contain one or more naturally-occurring amino acid derivatives ofthe twenty standard amino acids. For example: 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Proteins or polypeptides of the present invention also include anypolypeptide having one or more additions and/or deletions or residuesrelative to the sequence of a polypeptide whose sequence is encoded inthe DNA of gp100.

In one embodiment of the invention, gp100 epitopes are determined andbased upon a sequence motif. In order to generate immunogenic peptidesfor induction of a CD4+ T cell response, a variety of peptide epitopeswere synthesized in which at least one amino acid position was changedbased on the binding motifs of peptides presented by HLA-DRβ*0701 (Chiczet al., J. Exp. Med. 178:27-47, 1993; Rammensee, H. G. Immunogenetics41:178-228, 1995). Amino acids at precise positions in the gp100 peptidesequence have been suggested as anchor residues to the MHC molecule.Also, the amino acid sequence in the groove of the MHC moleculecorresponding to the contact zone with the peptide influences thecomposition of the anchor residues. MHC molecules are highly polymorphicin the population. The anchor residue motifs differ depending on the MHCproteins expressed.

Optimal anchor residues have been identified by sequencing severalpeptides bound to common MHC class I and class II molecules. Forexample, starting with the first amino acid bound to MHC-DRβ1*0701,anchor residues have been identified at position 1, 4, 6 and 9.Frequently, more than one amino acid with similar properties (charge,hydrophobicity, etc.) can be found for a particular anchor residue.However, these putative optimal anchor residues are not perfect andoften, exceptions can be seen. Modifications according to the optimaldefined binding motif can be made at putative anchor residues of apeptide in order to increase the affinity to the MHC class II moleculeand its immunogenicity. The consensus motif of the present inventioncomprises: position 1 having a residue comprising Phe, Tyr, Trp, Ile,Leu, or Val; position 4 having a residue comprising Asp, Glu, His, Lys,Asn, Gln, Arg, Ser, Thr, or Tyr; position 6 having a residue comprisingAsn, Ser, or Thr; and position 9 having a residue comprising Val, Ile,Leu, Tyr, or Phe, or derivatives thereof.

This invention also provides a recombinant DNA molecule comprising allor part of the gp100 nucleic acid sequence and a vector. Expressionvectors suitable for use in the present invention comprise at least oneexpression control element operationally linked to the nucleic acidsequence. The expression control elements are inserted in the vector tocontrol and regulate the expression of the nucleic acid sequence.Expression control elements and operational elements necessary orpreferred for appropriate transcription and subsequent translation areused.

It will be understood by one skilled in the art that the correctcombination of required or preferred expression control elements willdepend on the host system chosen. It will further be understood that theexpression vector should contain additional elements necessary for thetransfer and subsequent replication of the expression vector containingthe nucleic acid sequence in the host system. It is also understood thatthe skilled artisan will be able to easily construct such vectors usingconventional methods (Ausubel et al., (1987) in “Current Protocols inMolecular Biology”, John Wiley and Sons, New York, N. Y.) orcommercially available vectors. The host organism into which arecombinant expression vector containing all or part of the gp100nucleic acid sequence has been inserted and the means by which thevector carrying the gene of interest is introduced into the cell arecommonly known in the art (Sambrook et al., (1989) in “MolecularCloning. A Laboratory Manual”, Cold Spring Harbor Press, Plainview, N.Y.).

More specifically, a viral vector, such as a retroviral vector, can beintroduced into mammalian cells. Examples of mammalian cells into whichthe retroviral vector can be introduced include, but are not limited to,primary mammalian cultures or continuous mammalian cultures, COS cells,NIH3T3, or 293 cells (ATTC #CRL 1573). The means by which the vectorcarrying the gene may be introduced into a cell includes, but is notlimited to, microinjection, electroporation, transfection ortransfection using DEAE dextran, lipofection, calcium phosphate or otherprocedures known to one skilled in the art (Sambrook et al. (EDS) (1989)in “Molecular Cloning. A laboratory manual”, Cold Spring Harbor PressPlainview, N. Y.).

Another aspect of this invention relates to a host organism into whichrecombinant expression vector containing all or part of the gp100nucleic acid sequence has been inserted. The host cells transformed withthe gp100 nucleic acid sequence of this invention includes eukaryotes,such as animal, plant, insect and yeast cells and prokaryotes, such asE. coli. The means by which the vector carrying the gene may beintroduced into the cell includes, but is not limited to,microinjection, electroporation, transduction, or transfection usingDEAE-dextran, lipofection, calcium phosphate or other procedures knownto one skilled in the art (Sambrook et al. (1989) in “Molecular Cloning.A Laboratory Manual”, Cold Spring Harbor Press, Plainview, N. Y.).

“Melanoma antigen” or “immunogen” means all or part thereof of the gp100protein or peptides based on the gp100 protein sequence capable ofcausing a cellular or humoral immune response in a mammal. Such antigensare also reactive with antibodies from animals immunized with all, partor parts of the gp100 protein.

In one embodiment of the invention, a composition that can induce animmune response or an immunogen is prepared using recombinant gp100protein or peptide expression vectors. To provide animmune-response-inducing composition or an immunogen to an individual, agenetic sequence which encodes for all or part of the gp100 nucleic acidsequence is inserted into an expression vector, as described above, andintroduced into the mammal to be immunized. Examples of vectors that maybe used in the aforementioned immune-response-inducing compositions,such as vaccines, include, but are not limited to, defective retroviralvectors, adenoviral vectors, vaccinia viral vectors, fowl pox viralvectors, or other viral vectors (Mulligan, R. C., (1993) Science260:926-932).

The immune-response-inducing composition or immunogen can beadministered in accordance with conventional methods, alone or incombination with adoptive immunotherapy, e.g., T-cells generated againstthe gp100 epitope. For example, the immunogen can be used in a suitablediluent, such as saline or water, or complete or incomplete adjuvants.Further, the immunogen can be bound to a carrier to make the proteinimmunogenic. The immunogen also can be coupled with lipoproteins oradministered in liposomal form or with adjuvants. The immunogen can beadministered by any route, such as intravenous, intraperitoneal,intramuscular, subcutaneous, and the like. The immunogen can beadministered once or at periodic intervals until a significant titer ofanti-gp100 immune cells or anti-gp100 antibody is produced. The presenceof anti-gp100 immune cells can be assessed by measuring the frequency ofprecursor CTL (cytotoxic T-lymphocytes) or CD4+ T cells against gp100antigen prior to and after immunization by a CTL precursor analysisassay (Coulie, P. et al., (1992) International Journal Of Cancer50:289-297). Antibodies can be detected in the serum using theimmunoassay described above.

In yet another embodiment of this invention, multivalentimmune-response-inducing compositions, e.g., vaccines, or immunogensagainst one or more melanoma antigens are provided. Such multivalentcompositions can comprise all or part of the gp100 protein peptides oranalogs, or combinations thereof.

In a further embodiment of this invention, the recombinant or naturalgp100 protein, peptides, or analogs thereof, and/or pharmaceuticalcompositions or formulations comprising the recombinant or natural gp100protein, peptides, or analogs thereof, can be used as animmune-response-inducing composition or an immunogen in a therapeutic orpreventative manner. When provided prophylactically, theimmune-response-inducing composition or immunogen is provided in advanceof any evidence of melanoma. The prophylactic administration of thegp100 immune-response-inducing composition or immunogen serves toinhibit, prevent or attenuate melanoma in a mammal. In a preferredembodiment, a method of treating a cancer, such as melanoma, in a mammalcomprising administering an immune-response-inducing composition orimmunogen having one or more gp100 peptides. Theimmune-response-inducing composition or immunogen can be provided as apharmaceutical composition comprising an immunogenic peptide having acontiguous gp100 amino acid sequence of 40 amino acids or less,comprising MLGTHTMEVTV (SEQ ID NO:1) or a variant thereof, or any of SEQID NOs:2-21, in an amount effective to stimulate or induce animmunotherapeutic response. Additionally, a method of inhibiting orpreventing a recurrence of, for example, melanoma cancer in a mammal byadministering an immune-response-inducing composition or immunogencomprising one or more gp100 peptides. Preferably, the immunogen isprovided as a pharmaceutical composition comprising an immunogenicpeptide having a contiguous gp100 amino acid sequence of 40 amino acidsor less, comprising MLGTHTMEVTV (SEQ ID NO:1) or a variant thereof, orany of SEQ ID NOs:2-21, or variants thereof, in an amount effective tostimulate or induce an immunoprophylactic response.

More specifically, when provided prophylactically, the immunogen isprovided in advance of any evidence or in advance of any symptom due tomelanoma or after surgical resection of a tumor to prevent recurrence.Preferably, the immunogen is prophylactically administered in aneffective amount ranging from about 0.01-100 mg per patient per dose.The prophylactic administration of the immunogen serves to inhibito,prevent or attenuate melanoma in a mammal. Examples of such mammalsinclude, but are not limited to, humans with a family history ofmelanoma, humans with a history of atypical moles, humans with a historyof FAM-M syndrome or humans afflicted with melanoma previously resectedand, therefore, at risk for reoccurrence.

In yet another embodiment, the immune-response-inducing composition,e.g., vaccine, or immunogen, when provided therapeutically, is providedto enhance the patient's own immune response to the tumor antigenpresent on the melanoma or metastatic melanoma. Theimmune-response-inducing composition, which acts as an immunogen, may beobtained from various sources, including, for example, a cell, a celllysate from cells transfected with a recombinant expression vector, acell lysate from cells transfected with a gp100 recombinant expressionvector, or a culture supernatant containing the expressed protein.Alternatively, the immunogen may be a partially or substantiallypurified gp100 protein, peptide or analog thereof produced naturally,recombinantly, or synthetically. The proteins or peptides may beconjugated with lipoprotein or administered in liposomal form or withadjuvant. Further, the immunogen may be provided at (or shortly after)the onset of the disease or at the onset of any symptom of the disease.Preferably, the immunogen is therapeutically administered in aneffective amount ranging from 0.01 to 100 mg per patient per dose. Thetherapeutic administration of the immunogen serves to attenuate thedisease. In a preferred embodiment of the invention, the gp100immunogenic peptides may be used for the therapeutic and adjuvant(prevention of recurrent cancers) treatment of melanomas and are notlimited to subjects expressing the MHC Class II molecule DRβ*0701, asClass II-restricted peptides are often capable of binding to more thanone Class II molecule (Chicz et al. J. Exp. Med. 178:27-47, 1993;Malcherek et al. J. Exp. Med. 181:527-536, 1995).

TABLE 1 Gp100 Peptide Identification (IFN-γ; pg/ml) SEQ ID gp100 50 μM10 μM NO: 170-190     LSIG TGRAMLG THTMEVTVYH >25,000 21,000 3 168-188  SGLSIG TGRAMLG THTMEVTV 4,200 1,655 14 166-186 PVSGLSIG TGRAMLG THTMEV19 <8 20 172-192       IG TGRAMLG THTMEVTVYH RR >25,000 17,050 4 174-194TGRAMLG THTMEVTVYH RRGS >25,000 17,140 5 176-196   RAMLG THTMEVTVYHRRGSRS 15,230 912 15 178-198     MLG THTMEVTVYH RRGSRSYV 5,940 52 16TGRAMLG THTMEVTVYH >25,000 14,010 2 TGRAMLG THTMEVTVYH R >25,000 11,1006 TGRAMLG THTMEVTVYH RR >25,000 11,550 7 TGRAMLG TNTMEVTVYH RRG >25,00017,910 8        G TGRAMLG THTMEVTVYH RRG >25,000 22,910 9        GTGRAMLG THTMEVTVYH >25,000 18,770 10       IG TGRAMLG THTMEVTVYH >25,00017,590 11      SIG TGRAMLG THTMEVTVYH >25,000 20,710 12      SIG TGRAMLGTHTMEVTVYH R >25,000 12,210 13 TGRAFLG THTMEVTVYH RRGS >25,000 19,390 17TGRALLG THTMEVTVYH RRGS >25,000 23,140 18 TGRAYLG THTMEVTVYHRRGS >25,000 11,640 19 un-pulsed <8 NY-ESO-1 (0.14 μm) <8 gp100 (0.14μm) 13,730

In addition to use as an immune-response-inducing composition, e.g.,vaccine, or an immunogen of the invention, the pharmaceuticalcompositions can be used to prepare antibodies to gp100 antigen,peptides or analogs thereof. The antibodies can be used directly asanti-melanoma agents. To prepare antibodies, a host animal is immunizedusing the gp100 protein, peptides or analogs thereof as the immunogenand bound to a carrier as described above for immune-response-inducingcompositions. The host serum or plasma is collected following anappropriate time interval to provide a composition comprising antibodiesreactive with the immunogen as is understood by the skilled artisan. Thegamma globulin fraction or the IgG antibodies can be obtained, forexample, by using saturated ammonium sulfate or DEAE Sephadex, or othertechniques known to those skilled in the art. The antibodies aresubstantially free of many of the adverse side effects which may beassociated with other anti-cancer agents such as chemotherapy.

Immunoassays of the present invention may be radioimmunoassay, Westernblot assay, immunofluorescent assay, enzyme immunoassay,chemiluminescent assay, immunohistochemical assay and the like. (In“Principles and Practice of Immunoassay” (1991) Christopher P. Price andDavid J. Neoman (eds), Stockton Press, New York, N. Y.; Ausubel et al.(eds) (1987) in “Current Protocols in Molecular Biology” John Wiley andSons, New York, N. Y.). Standard techniques known in the art for ELISAare described in Methods in Immunodiagnosis, 2nd Edition, Rose andBigazzi, eds., John Wiley and Sons, New York 1980 and Campbell et al.,Methods of Immunology, W. A. Benjamin, Inc., 1964, both of which areincorporated herein by reference. Such assays may be direct, indirect,competitive, or noncompetitive immunoassays as described in the art (In“Principles and Practice of Immunoassay” (1991) Christopher P. Price andDavid J. Neoman (eds), Stockton Press, NY, N. Y.; Oellirich, M. 1984. J.Clin. Chem. Clin. Biochem. 22: 895-904). Biological samples appropriatefor such detection assays include mammalian tissues, melanoma andmelanocyte cell lines, skin, retina, lymph nodes, pathology specimens,necropsy specimens, and biopsy specimens. Proteins may be isolated frombiological samples by conventional methods (see, e.g., Ausubel et al.,(eds) (1987) in “Current Protocols in Molecular Biology” John Wiley andSons, New York, N. Y.).

One skilled in the art will understand that the bioassays orimmunoassays of the present invention may be used in the analysis ofbiological samples or tissues from any vertebrate species. In apreferred embodiment, mammalian biological samples or tissues areanalyzed. Tissue includes, but is not limited to, single cells, wholeorgans and portions thereof. Biological samples include, but are notlimited to, tissues, primary mammalian cultures, continuous mammaliancell lines, such as melanocyte cell lines, mammalian organs, such asskin or retina, tissues, biopsy specimens, melanoma and lymph nodebiopsy samples, neoplasms, pathology specimens, and necropsy specimens.Mammal includes but is not limited to, humans, monkeys, dogs, cats,mice, rats, pigs, cows, pigs, horses, sheep and goats.

Examples of diseases that can be assessed by these immunoassays,include, but are not limited to, melanomas and tissues which aresecondary sites for melanoma metastasis. By alteration in level ofexpression, we mean an increase or decrease of the gp100 protein orportions thereof relative to a control sample. Alteration is also meantto encompass substitution, deletion or addition mutants of the gp100protein. Such mutations can be determined by using antibodies known toreact with specific epitopes of the gp100 protein and determining whichepitopes are present relative to a control. These antibodies cantherefore be used in an immunoassay to diagnose, assess or prognose amammal afflicted with the disease.

In a preferred embodiment, the gp100 antibodies are used to assess thepresence of the gp100 antigen from a tissue biopsy of a mammal afflictedwith melanoma using immunocytochemistry. Such assessment of thedelineation of the gp100 antigen in a diseased tissue may be used toprognose the progression of the disease in a mammal afflicted with thedisease. Conventional methods for immunohistochemistry are described inHarlow and Lane (eds) (1988) In “Antibodies A Laboratory Manual”, ColdSpinning Harbor Press, Cold Spring Harbor, N. Y.; Ausbel et al. (eds)(1987). In Current Protocols In Molecular Biology, John Wiley and Sons(New York, N. Y.).

In yet a further embodiment, the recombinant protein expressed by hostcells can be obtained as a crude lysate or can be purified by standardprotein purification procedures known in the art which may includedifferential precipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. (Ausubel et. al., (1987) in“Current Protocols in Molecular Biology” John Wiley and Sons, New York,N. Y.). Antibodies of this invention can be used to purify the gp100protein or portions thereof. In the case of immunoaffinitychromatography, the recombinant protein can be purified by passagethrough a column containing a resin which has bound thereto antibodiesspecific for the gp100 protein (Ausubel et. al., (1987) in “CurrentProtocols in Molecular Biology” John Wiley and Sons, New York, N. Y.). Apreferred method of purifying gp100 protein can be performed byelectrophoresing, collecting, dialyzing, and precipitating the gp100protein, such that a purity of more than 80% was produced (see Example10).

The antibodies also can be used as a means of enhancing the immuneresponse. The antibodies can be administered in amounts similar to thoseused for other therapeutic administrations of antibody. For example,pooled gamma globulin is administered at a range of about 1-100 mg perpatient. Thus, antibodies reactive with the gp100 antigen can bepassively administered alone or in conjunction with other anti-cancertherapies to a mammal afflicted with melanoma. Examples of anti-cancertherapies include, but are not limited to, chemotherapy, radiationtherapy, and adoptive immunotherapy therapy with TIL.

The antibodies or chimeric antibodies described herein also can becoupled to toxin molecules, radioisotopes, and drugs by conventionalmethods (Vitetta et al. (1991) in “Biologic Therapy of Cancer” De Vita VT, Hellman S., Rosenberg, S. A. (eds) J. B. Lippincott Co. Philadelphia;and Larson, S. M. et al. (1991) in “Biological Therapy of Cancer” DeVita V. T., Hellman S., Rosenberg, S. A. (eds) J. B. Lippincott Co.,Philadelphia). Examples of toxins to which the antibodies can be coupledto include, but are not limited to, ricin or diphtheria toxin. Examplesof drugs or chemotherapeutic agents include, but are not limited to,cyclophosphamide or doxorubcin. Examples of radioisotopes, include, butare not limited to, ¹³¹I. Antibodies covalently conjugated to theaforementioned agents can be used in cancer immunotherapy for treatingmelanoma.

The antiserum from immunized individuals can be administered as aprophylactic measure for individuals who are at risk of developingmelanoma. By “prophylactic” and “prophylaxis” is meant the inhibition orprevention of melanoma. One of ordinary skill in the art readilyappreciates that, while complete prevention is desired, any degree ofinhibition is can be beneficial. The antiserum is also useful intreating an individual afflicted with melanoma for post-diseaseprophylaxis.

The gp100 derived proteins and/or peptides of the invention are alsointended for use in producing antiserum designed for pre- orpost-disease prophylaxis. Here the gp100 antigen, peptides or analogsthereof is formulated with a suitable adjuvant and administered byinjection to human volunteers, according to known methods for producinghuman antisera. Antibody response to the injected proteins is monitored,during a several-week period following immunization, by periodic serumsampling to detect the presence of anti-gp100 serum antibodies, using animmunoassay as described herein.

This invention describes a method of retrovirally transducing dendriticcells (DCs) with the melanoma differentiation antigen gp100, which arethen stimulated. The gp100-transduced DCs generated T cells thatrecognized three distinct HLA-A2 restricted epitopes of the tumorantigen. CD4+ helper T cells specific to a new HLA-DRβ1*0701 epitope ofgp100 were generated. The present invention allowed the production ofretrovirally-transduced antigen presenting cells (APCs), such as forexample, dendritic cells (DCs), to generate T cells reactive againstmultiple MHC class I and class II epitopes of a tumor antigen which canplay a significant role in analysis of tumor antigens and moreimportantly, for cancer patient immunotherapy. In so doing, a strongimmune response against the gp100 antigen was generated.

Additionally provided is a method of transducing APCs with a gp100nucleic acid sequence comprising: obtaining T cells, for example, CD34+cells; culturing the T cells with growth factor to stimulatedifferentiation and proliferation resulting in T cells, such as forexample, DCs; and introducing expression vectors, preferably, but notlimited to, retroviral vectors and the like, containing all or one ormore parts of the gp100 gene, wherein the gp100 gene of interest isexpressed. Examples of cells that may be used to deliver nucleic acidsencoding gp100 antigens of the present invention include, but are notlimited to, T2 cells, (Cerundolo, V. et al. (1990) Nature, 345: 449-452)or EBV transformed B cell lines (Topalian et al. (1989) J. Immunol. 142:3714-3725).

In yet another embodiment of this invention, all, part, or parts of thegp100 protein or gp100 peptides can be exposed to dendritic cells (DCs)cultured in vitro. The DCs should be exposed to antigen for sufficienttime to allow the antigens to be internalized and presented on the DCssurface (see Example 9). The resulting DCs or the dendritic cell processantigens then can be administered to an individual in need of therapy.In so doing, the cultured DCs provide a means of producing Tcell-dependent antigens comprised of dendritic cell-modified antigen orDCs pulsed with antigen, in which the antigen is processed and expressedon the antigen loaded-dendritic cell. The gp100 antigen-loaded DCs maybe used as immunogens for vaccines or for the treatment of melanoma.Such methods are described in Steinman et al. (WO93/208185) and inBanchereau et al. (EPO Application 0563485A1), which are incorporatedherein by reference.

In yet another embodiment of this invention, T cells isolated fromindividuals can be exposed to the gp100 protein or portions thereof invitro and then administered to a patient in need of such treatment in atherapeutically effective amount. Examples of where T-lymphocytes usefulin the invention are, include but are not limited to, peripheral bloodcell lymphocytes (PBL), lymph nodes, or tumor infiltrating lymphocytes(TIL). Such lymphocytes can be isolated from the individual to betreated or from a donor by methods known in the art and cultured invitro (Kawakami, Y. et al. (1989) J. Immunol. 142: 2453-3461).Lymphocytes are cultured in media such as RPMI or RPMI 1640 or AIM V for1-10 weeks. Viability is assessed by trypan blue dye exclusion assay.The lymphocytes are exposed to all or part of the gp100 protein for partor all of the culture duration.

In another embodiment, all or parts thereof of a substantially orpartially purified gp100 protein and/or gp100 peptide can beadministered as an immunogen in a pharmaceutically- andphysiologically-acceptable carrier. Ranges of gp100 protein to beadministered are 0.001 to 100 mg per patient; preferred doses are 0.01to 100 mg per patient. Immunization is repeated as necessary, until asufficient titer of anti-immunogen antibody or immune cells has beenobtained. While it is possible for the immunogen to be administered in apure or substantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation.

In a preferred embodiment, the lymphocytes are exposed to the gp100peptide of this invention at a concentration of 1-10 micrograms (μg)/mlper 10⁷ cells for all or part of the duration of lymphocyte culture.Peptides can be administered either alone or conjugated. After beingsensitized to the peptide, the T lymphocytes are administered to themammal in need of such treatment. Examples of how these sensitized Tcells can be administered to the mammal include, but are not limited to,intravenously, intraperitoneally or intralesionally. Parameters that canbe assessed to determine the efficacy of these sensitized T-lymphocytesinclude, but are not limited to, production of immune cells in themammal being treated or tumor regression. Conventional methods are usedto assess these parameters. Such treatment can be given in conjunctionwith cytokines or gene modified cells (Rosenberg, S. A., et al. (1992)Human Gene Therapy, 3: 75-90; Rosenberg, S. A., et al. (1992) Human GeneTherapy, 3: 57-73).

Mammalian cells expressing the gp100 antigen and/or gp100 peptides canbe administered to mammals and serve as an immune-response-inducingcomposition, e.g., vaccine, or immunogen. Examples of how the cellsexpressing gp100 antigens or peptides can be administered include, butis not limited to, intravenous, intraperitoneal or intralesional. In apreferred embodiment, the entire gp100 nucleic acid sequence is insertedinto the gp100 expression vector and introduced into the mammaliancells.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it aspharmaceutical compositions, formulations or preparations as describedabove for gp100. Conventional methods can be used to administer theimmunogen or immune-response-inducing composition, e.g., vaccine, aspreviously described above for gp100. The gp100 immunogenic peptides andnucleic acids sequences encoding them can be used in bioassays, or togenerate antibodies.

The viral vectors carrying all or part of the gp100 nucleic sequence canbe introduced into a mammal either prior to any evidence of melanoma tomediate regression of the disease in a mammal afflicted with melanoma orto prevent the recurrence of disease. Examples of methods foradministering the viral vector into the mammals include, but are notlimited to, exposure of cells to the virus ex vivo, or injection of theretrovirus or a producer cell line of the virus into the affected tissueor intravenous administration of the virus. For a detailed descriptionof ex vivo gene therapy, see U.S. Pat. No. 5,399,346.

Alternatively, the viral vector carrying all or part of the gp100nucleic acid sequence can be administered locally by direct injectioninto the melanoma lesion or topical application in a pharmaceuticallyacceptable carrier. The quantity of viral vector, carrying all or partof the gp100 nucleic acid sequence, to be administered is based on thetiter of virus particles. A preferred range of the immunogen to beadministered is 10⁶ to 10¹¹ virus particles per mammal, preferably ahuman. After immunization, the efficacy of the vaccine or immunogen canbe assessed by production of antibodies or immune cells that recognizethe antigen, as assessed by specific lytic activity or specific cytokineproduction or by tumor regression. One skilled in the art is aware ofconventional methods to assess the aforementioned parameters. If themammal to be immunized is already afflicted with melanoma or metastaticmelanoma, the vaccine can be administered in conjunction with othertherapeutic treatments. Examples of other therapeutic treatmentsinclude, but are not limited to, adoptive T cell immunotherapy,coadministration of cytokines, or other therapeutic drugs for melanoma.

The pharmaceutical compositions or formulations of the presentinvention, both for veterinary and for human use, comprise a gp100protein, gp100 peptide, and/or immunogen as described herein, togetherwith one or more pharmaceutically acceptable carriers and, optionally,other therapeutic ingredients. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Theformulations can conveniently be presented in unit dosage form and canbe prepared by any method well known in the pharmaceutical art. Carrierscan also include, but are not limited to excipients and/or diluents.

In a preferred embodiment of the invention, a vaccine or immunogenicformulation comprising an immunogen that induces an immune responsedirected against the melanoma associated antigen, gp100, is provided.The gp100 formulations are evaluated first in animal models, initiallyrodents, and in nonhuman primates and then in humans. The safety of theimmunization procedures is determined by looking for the effect ofimmunization on the general health of the immunized animal (weightchange, fever, appetite behavior, etc.) and looking for pathologicalchanges on autopsies. After initial testing in animals, melanoma cancerpatients are tested. Conventional methods are used to evaluate theimmune response of the patient to determine the efficiency of thevaccine.

The formulations of the present invention can incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids which can be used eitheron their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the immunogen of the present invention, anti-adsorptionagent may be used.

When oral preparations are desired, the compositions can be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

All methods include the step of bringing into association the activeingredient with the carrier, which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions which are preferablyisotonic with the blood of the recipient. Such formulations can beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances, such as sodiumchloride (e.g. 0.1-2.0M), glycine, and the like, and having a bufferedpH compatible with physiological conditions to produce an aqueoussolution, and rendering said solution sterile. These can be present inunit or multi-dose containers, for example, sealed ampoules or vials.

Additional pharmaceutical methods can be employed to control theduration of action. Controlled release preparations can be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery can be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate the gp100protein, peptides and analogs thereof into particles of a polymericmaterial, such as polyesters, polyamino acids, hydrogels, poly(lacticacid) or ethylene vinylacetate copolymers. Alternatively, instead ofincorporating these agents into polymeric particles, it is possible toentrap these materials in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxy-methylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

Local administration to the afflicted site can be accomplished throughmeans known in the art, including, but not limited to, topicalapplication, injection, and implantation of a porous device containingcells recombinantly expressing the infusion, implantation of a porousdevice in which the gp100 antibodies or chimeric antibodies, antibodiescoupled to toxins, drugs or radiolabels or portions thereof arecontained.

When used as a means of inducing gp100 antibodies in an animal, themanner of injecting the antibody is the same as for vaccinationpurposes, namely intramuscularly, intraperitoneally, subcutaneously,interlesionally, or the like in an effective concentration in aphysiologically suitable diluent with or without adjuvant. One or morebooster injections may be desirable.

The proteins of the present invention can be supplied in the form of akit, alone, or in the form of a pharmaceutical composition as describedabove.

The above described antibodies and antigen-binding fragments thereof canbe supplied in kit form alone, or as a pharmaceutical composition for invivo use. The antibodies can be used for therapeutic uses, diagnosticuse in immunoassays, or as an immunoaffinity agent to purify the gp100protein or peptides as described herein.

All books, articles, and patents referenced herein are incorporated byreference in toto. The following examples illustrate various aspects ofthe invention and in no way intended to limit the scope thereof.

Example 1 Media and Cell Culture

Complete medium consisted of Iscove's (Biofluids Inc.; Rockville, Md.and Life Technologies; Gaithersburg, Md.) supplemented with 10% human ABserum (male, heat inactivated; Gemini Bio-Products, Calabasas, Calif.),1 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin (all fromBiofluids Inc.) and 50 μg/ml gentamicin (Life Technologies). DC mediumconsisted of complete medium supplemented with 100 ng/ml of GM-CSF, 100ng/ml of TNF-α (both from Peprotech, Rocky Hill, N.J.) and 40 ng/ml ofstem cell factor (SCF; R&D System Inc., Minneapolis, Minn.). T cellmedium consisted of complete medium supplemented with 60 to 300 IU/mlrIL-2 (Chiron; Emeryville, Calif.). T cell clones and TIL 1520 werecultured in AIM-V medium (Life Technology) supplemented with 5% humanserum and 1 mM glutamine, 50 U/ml penicillin, 50 μg/ml, streptomycin and50 μg/ml gentamicin. rIL-2 at a concentration 6,000 IU/ml was added forTIL 1520 and 300 IU/ml for the T cell clones.

The melanoma cell line from patient A was developed at the SurgeryBranch NCI/NIH; (Topalian et al. Proc. Natl. Acad. Sci. USA. 91:9461-65,1994). Since the melanoma line from patient A failed to express gp100,this line was transduced using a VSV-pseudotyped retroviral vectorexpressing either gp100 or green fluorescent protein (GFP).EBV-immortalized B cells were generated as previously described(Topalian et al. Proc. Natl. Acad. Sci. USA. 91:9461-65, 1994).Transduced cells were cultured in RPMI-based medium containing 500 μg/mlof geneticin sulfate (Life Technologies). All tumor and EBV-B cell lineswere grown in RPMI 1640 medium (Life Technologies) supplemented with 10%heat inactivated FBS (Biofluids Inc. and Life Technologies) andantibiotics.

CD34⁺ hematopoietic progenitor cells (HPC) were obtained from HLA-A2positive patients undergoing treatment for melanoma as part of anInstitutional Review Board-approved protocol (Surgery Branch, NCI).CD34⁺ cells were mobilized in peripheral blood by five daily s.c.injections of 10 μg/kg granulocyte colony-stimulating factor (Neupogen,Amgen, Thousand Oaks, Calif.), followed by a lymphocytopheresis toobtain PBMCs on day 6. CD34⁺ cells were selected by an immunoaffinitycolumn (CellPro Inc., Bothed, Wash.) and cryopreserved. A singleleukapheresis typically yielded 2-5×10⁸ CD34⁺ cells.

Example 2 Retroviral Vectors

The PG13-based gp100 retroviral packaging cell line was generated byinserting the complete gp100 cDNA into the retroviral vector SAMEN(Kershaw et al. Hum. Gene Ther. 11:2445-52, 2000) as describedpreviously (Reeves et al. Cancer Res. 56:5672-5677, 1996). Producer cellmedium consisted of Dulbecco's Modified Eagle Medium (Life Technologies)supplemented with 10% heat inactivated FBS and antibiotics.

The gp100-VSV-pseudotyped retroviral system was prepared by firstinserting the complete gp100 sequence in the pCLNC retroviral plasmid(Naviaux et al. J. Virol. 70:5701-5705, 1996)(28). The pCLNC-gp100 andpMDG-VSV plasmids were co-transfected in 293-gag-pol packaging cellsusing Lipofectamine Plus (Life Technologies). The 293-gag-pol packagingcells (Salk Institute; La Jolla, Calif.), were cultured in DMEMsupplemented with 10% heat inactivated FBS and antibiotics. Medium waschanged 16 hours and 48 hours after transfection. Culture supernatantswere harvested on days 3 and 4 after transfection of the 293-gag-polcells. Producer cells were removed from retroviral supernatant byfiltration with 0.2 μm filter (Nalgene; Rochester, N. Y.). Supernatantswere immediately frozen at −70° C. for future use.

Example 3 Transduction of CD34+ Derived Dendritic Cells

CD34⁺ cells were differentiated to dendritic cells according to theprotocol previously described (Reeves et al. Cancer Res. 56:5672-5677,1996). Briefly, CD34⁺ cells were thawed, washed in complete medium andplated at 5×10⁶ cells/well in a 6 well plate or 5×10⁵ cells/well in a 24well plate in complete DC medium containing TNF-α, SCF and GM-CSF.Cultured cells were harvested on days 5 and 10, centrifuged andresuspended in complete DC medium with cytokines. Cells were utilized onday 14 and DC phenotype was confirmed by morphological and FACS analyses(Reeves et al. Cancer Res. 56:5672-5677, 1996).

For transduction with the PG13 system, CD34⁺ cells were co-cultured withirradiated PG13-SAM-gp100-EN producer cells (31 Gy; 7×10⁵ cells/well in6 well plate) in complete DC medium with cytokines containing 8 μg/ml ofpolybrene (Aldrich Chemical Co., Milwaukee, Wis.). After 30 h, DCs werereplated on fresh irradiated producer cells in DC medium includingcytokines without polybrene for 24 h. On day 3, transduced DCs wereresuspended in fresh complete DC medium in a 6 well plate and thedifferentiation was completed as described earlier.

For transduction with the VSV-pseudotyped retroviruses, retroviralsupernatant was added to cultured CD34⁺ cells on days 2 and 3 at a ratioof 1:1 with culture medium. GM-CSF, SCF, TNF-γ and polybrene were addedand cells were spun in the plate at 1,000×g for one hour. On day 4,transduced DCs were resuspended in fresh complete DC medium in a 6 wellplate and the differentiation was completed as described above.

Example 4 Stimulation of Autologous T Lymphocytes with TransducedDendritic Cells

Autologous human PBMC (peripheral blood mononuclear cells) were obtainedfrom leukapheresis of patients at the Surgery Branch (NCI) andcryopreserved for use in experiments. T lymphocytes were isolated fromPBMC using a human T cell immunoaffinity column (R&D Inc). Purified Tcells (2×10⁶) were co-cultured with irradiated gp100-transduced DCs(2×10⁵, 15 Gy) in a 24 well plate in 2 ml of complete medium withoutrIL-2. rIL-2 (60 to 300 U/ml) was added on day 2 and the cells werediluted with fresh complete medium and rIL-2 to keep the cellconcentration at 1-2×10⁶ cell/ml. T cells from patient A were obtainedprior to any non-surgical treatment of melanoma and were stimulated oncewith gp100-transduced DCs. T lymphocytes from patient B were obtainedeight months after immunization with gp100₂₈₀₋₂₈₈ peptide in incompleteFreund's adjuvant (Salgaller et al. Cancer Res. 56:4749-4757, 1996). Insome cultures, a combination of soluble trimeric recombinant CD40L(Immunex Corporation, Seattle, Wash.) and lipopolysaccharide preparedfrom Salmonella typhimurium (Sigma Chemical Co., St-Louis, Mo.) wereadded to the T cell stimulation with the gp100-transduced DCs. CD40L andLPS can increase the capacity of DCs to stimulate the generation ofantigen-specific T cells (Lapointe et al. Eur. J. Immunol. 30:3291-3298,2000).

In addition to presentation of epitopes by MHC class I, transduced DCsmay present peptides in the context of MHC class II molecules. T cellstimulations were next performed to determine whether gp100-transducedDCs could generate antigen-specific CD4⁺ T cells recognizing gp100peptides presented by MHC class II. T cells from patient A werestimulated twice with DCs retrovirally transduced with gp100. StimulatedT cells were analyzed for their capacity to recognize a gp100 expressingautologous melanoma cell line in an MHC class II restricted fashion. Thecultured T cells were reactive against the autologous tumor lineexpressing HLA-DR and gp100 but not against gp100-negative melanomacells or autologous cultured B cells.

The reactive bulk T cell culture was cloned by limiting dilution andwells containing growing cells were screened using autologous melanomacells expressing gp100 or GFP as a negative control. The bulk T cellcultures were cloned by limiting dilution at 1, 2 or 10 cells/well in 96round bottom well plates (Dudley et al. J. Immunother. 22:288-298,1999). Cloning was done in complete medium using AIM-V mediumsupplemented with 5% human AB serum, 1 mM glutamine, 50 U/ml penicillin,50 μg/ml streptomycin and 50 μg/ml gentamicin in the presence of 5×10⁴irradiated PBMC prepared from 3 different donors (from normal volunteersat the Clinical Center, NIH), 30 ng/ml of anti-CD3 (OKT3; Ortho-Biotech,Raritan, N.J.) and 300 U/ml of rIL-2. Relevant clones were expanded inT-25 tissue culture flasks at 1 to 5×10⁵ cells/flask with 2.5×10⁷irradiated PBMC feeders prepared from 3 different donors (Blood bank,Clinical center, NIH) and 30 ng/ml of anti-CD3 in 25 ml of AIM-Vcomplete medium. On day 2, 300 U/ml of rIL-2 was added and on day 5, 20ml of medium was replaced with fresh AIM-V complete medium containing300 U/ml of rIL-2. On day 8, cells were counted and the cellconcentration was maintained at 1 to 2×10⁶ cells/ml in AIM-V completemedium with 300 U/ml of rIL-2.

Forty-six of 167 wells were found to be specifically reactive againstthe autologous melanoma expressing gp100 and class II. The reactivity ofnine representative positive clones is presented in FIG. 1A along with 4representative negative wells (neg1 to neg4). Eight of the 46 positiveclones were expanded and further characterized. All 8 clones were CD4⁺suggesting recognition of gp100 peptides presented by MHC class II. Tofurther characterize the reactivity, some of the clones were co-culturedwith autologous B cells pulsed with a bacterially produced gp100protein. All the CD4⁺ T cell clones tested secreted IFN-γ when exposedto autologous B cells pulsed with gp100 but not those pulsed with anirrelevant tumor antigen prepared using a similar procedure. The resultsfrom two representative clones are presented in FIG. 1B. Clones 6 and 8failed to react against NY-ESO-1, a protein produced similarly to therecombinant gp100. In order to identify the MHC restriction element,antibodies known to bind and block the presentation by defined MHCmolecules were used. Recognition by clones 6 and 8 of target cellspulsed with the gp100 protein was inhibited when using blockingantibodies against MHC class II and HLA-DR but the recognition wasunchanged when using a blocking MHC class I antibody. Also, both clonesfailed to recognize gp100 when pulsed on un-matched HLA-DR B cells. Byusing HLA-DR matched B cells, the restriction element was identified asHLA-DRβ1*0701 (patient A is HLA-DRβ1*0701 and *1601; FIG. 2).

To find the epitope recognized, an overlapping gp100 peptide libraryconsisting of 68 peptides of 17 or 21 residues overlapping by 10-12amino acids was prepared. The autologous EBV-B cells were pulsed withthe peptide library and used as targets in a T cell recognition assayusing three different T cell clones. Two clones failed to recognize anypeptides and one clone specifically recognized peptide gp100₁₇₀₋₁₉₀(FIG. 3A; data from two clones shown). Using a gp100₁₇₀₋₁₉₀-specific Tcell clone, a titration experiment using the gp100₁₇₀₋₁₉₀ peptide pulsedon EBV-B cells revealed that the recognition was undetectable below 4-10μM (FIG. 3B). Interestingly, the gp100₁₇₀₋₁₉₀-specific T cell clonerecognized B cells pulsed with gp100 protein at a concentration as lowas 22 nM.

Finally, several peptides were synthesized in order to define an optimalHLA-DRβ1*0701 epitope (Rammensee, H. G. Immunogenetics 41:178-228, 1995;Chicz et al. J. Exp. Med. 178:27-47, 1993). As shown in table 1, nopeptides were better recognized at 10 or 1 μM compared with thegp100₁₇₀₋₁₉₀ peptide. Finally, substitutions were made at position 5 ofthe gp100₁₇₄₋₃₉₄ peptide presuming that this is position 1 according tothe defined motif for HLA-DRβ1*0701 (SYFPEITHI web site,http://syfpeithi.de/). However, none of the peptides were betterrecognized when pulsed at 10 μM or 1 μM compared to the wild typegp100₁₇₄₋₃₉₄ peptide.

The data presented demonstrate that CD34-derived dendritic cellsretrovirally-transduced with gp100 can generate T cells reactive againstMHC class II gp100 epitopes.

Example 5 T Cell Assays

The gp100 peptide library consisted of 68 peptides of 17 to 21 residuesoverlapping by 10 to 12 amino acids. The peptides were synthesized bysolid phase FMOC methodology as previously described (Parkhurst et al.J. Immunol. 157:2539-2548, 1996). A control peptide derived from the Igkappa chain known to bind to HLA-DRβ1*0701 was also prepared (Chicz etal. J. Exp. Med. 178:27-47, 1993). Recombinant gp100 protein was madeand purified as previously described Touloukian et al. J. Immunol.164:3535-3542, 2000). Recombinant NY-ESO-1 protein is another tumorantigen (Chen, Y T and L J Old. Cancer J. Sci. Am. 5:16-17, 1999) usedas negative control and was made and purified as previously described(Zeng et al. J. Immunol. 165:1153-1159, 2000). EBV-B cells (1×10⁵) werepulsed with gp100-purified protein or peptides for 18 hours in 96 wellflat-bottom plates and T cells were added directly to the pulsed-B cellsfor a 24 hours recognition assay. In some experiments, EBV-B cells(5×10⁵) were pulsed in 48 well plates for 18 hours and cells were washedtwice with PBS. T cells were then co-cultured with the pulsed EBV-Bcells in 96 well flat-bottom plates for 24 hours. Supernatants wereharvested and human IFN-γ was assayed by ELISA using a commerciallyavailable kit (R&D Inc.).

Example 6 IFN-γ Release Assay

Fifty to one hundred thousand responder cells and 4×10⁴-10⁵ stimulatorcells were mixed in 300 ul of AIM-V medium containing 1201 U/ml IL-2 perwell in a 96 flat-well microplate. Target cells (1×10⁵) were incubatedwith 1×10⁵ specific T cells in 250 μl of T cell medium (AIM-V-basedmedium) in 96 well flat-bottom plates. Supernatants were harvested after24 h and human IFN-γ was assayed by ELISA using a commercially availablekit (R&D, Inc.; Minneapolis, Minn.).

Example 7 Gp100 Immunogens as a Treatment for Melanoma in Mammals

Gp100 immunogens may be efficacious in treating mammals afflicted withmelanoma. For example, gp100 immune-response-inducing compositions maybe administered to individuals. Mammals are immunized with therecombinant proteins in the range of 1 mg-100 mg. Alternatively,patients, are immunized with the gp100 nucleic acid sequence insertedinto a viral vector such as vaccinia virus, adenovirus or fowl poxvirus. A range of about 10⁶-10¹¹ viral particles carrying the gp100nucleic acid sequences are administered per patient. The patients aremonitored for antibodies to the immunogen or increase in cytotoxiclymphocytes (CTL) recognizing the immunogen by conventional methods oralleviation of clinical signs and symptoms of the active disease.Specific parameters to be assessed include production of immune cellsthat recognize the vaccine antigen or tumor regression. Suchimmune-response-inducing compositions are administered eitherprophylactically or therapeutically. Alternatively, patients areimmunized with the gp100 nucleic acid sequence inserted into aretroviral vector. Suggested dose ranges of the antigen in retrovirusesare 10⁶-10¹¹ viral particles per patient. Response and efficacy of theretroviral vaccines are assessed as described above.

Example 8 Use of Lymphocytes Sensitized to Immunogenic Peptides Derivedfrom Melanoma Antigens for Therapeutically Treating Patients Afflictedwith Melanoma

T-lymphocytes presensitized to the melanoma antigen are effective intherapeutically treating patients afflicted with melanoma. TheT-lymphocytes are isolated from peripheral blood lymphocytes or tumorinfiltrating lymphocytes and exposed in vitro to the gp100 protein orpeptide. T-lymphocytes are isolated from peripheral blood or melanomatumor suspensions and cultured in vitro (Kawakami, Y. et al. (1988) J.Exp. Med. 168: 2183-2191). The T-lymphocytes are stimulated with 1 to 10μg/ml of the gp100-derived peptide and cultured for 2 to 6 weeks to geta high number of cells (Dudley, M. et al. (1999) J. Immunother.22:288-298). T-lymphocytes exposed to the antigen are administered tothe patient at about 10⁹-10¹² lymphocytes per patient. The lymphocytesmay be administered either intravenously, intraperitoneally orintralesionally. This treatment may be administered concurrently withother therapeutic treatments such as cytokines, radiotherapy, surgicalexcision of melanoma lesions, and chemotherapeutic drugs.

Example 9 Recombinant Human Gp100 Protein

The gene encoding human gp100 (h-gp100; Kawakami, et al. Proc. Natl.Acad. Sci. USA 91:6458, 1994) was amplified by PCR with primers(forward: 5′-AGGCGCAGACTTATGAAGCA-3′ (SEQ ID NO:24); reverse:5′-CTGCCCAAGGCCTGCTTCTTG-3′ (SEQ ID NO:25)) designed to delete theN-terminal 23 amino acids (probable signal sequence) and the C-terminal66 amino acids (probable transmembrane region). The truncated gene wasthen cloned into the PET28a⁺ expression vector (Novagen; Madison, Wis.)and transformed into BL21(DE3) Escherichia coli (Novagen). E. coli weregrown to OD600 0.6, then protein expression was induced with isopropylβ-D-thiogalactoside 1 μg/ml for 3 h. The bacteria were harvested;inclusion bodies were isolated and lysed in 6 M urea, then proteins werepurified by preparative scale SDS-PAGE in a Prep cell (Bio-Rad;Hercules, Calif.) electrophoresis chamber. Protein fractions at 61.5 kDa(theorectical molecular mass of truncated h-gp100) were collected,dialyzed, and precipitated, and a purity of more than 80% was estimatedbased on SDS-PAGE with Colloidal Blue (Novex; San Diego, Calif.)staining.

The present invention is not to be limited in scope by the nucleic acidsequences deposited, since the deposited embodiments is intended as asingle illustration of one aspect of the invention and any sequenceswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of thedependent claims. It is also to be understood that all base pair sizesgiven for nucleotides are approximated and are used for purpose ofdescription.

1. An immunogenic peptide consisting of a gp100 amino acid sequenceselected from SEQ ID NOs: 14-16 and 20-21, said peptide being reactivewith T cells or inducing an immune response.
 2. The immunogenic peptideof claim 1, wherein said peptide is recognized by HLA-DRβ1*0701restricted T cells.
 3. The immunogenic peptide of claim 1, wherein saidpeptide is selected from the group consisting of a native, a syntheticor a recombinant peptide.
 4. A pharmaceutical composition comprising animmunogenic peptide and a pharmaceutically-acceptable carrier therefor,said peptide consisting of a gp100 amino acid sequence selected from SEQID NOs: 14-16 and 20-21, said peptide being reactive with T cells.
 5. Amethod of transducing antigen presenting cells with a gp100 nucleic acidsequence encoding an immunogenic peptide consisting of an amino acidsequence selected from SEQ ID NOs: 14-16 and 20-21 comprising: a)obtaining T cells; b) introducing the gp100 nucleic acid sequence intothe antigen presenting cells; and c) stimulating the T cells.
 6. Amethod of determining immunogenicity of a peptide consisting of a gp100amino acid sequence selected from SEQ ID NOs: 14-16 and 20-21, saidpeptide being capable of reacting with T cells, comprising: a)incubating the peptide with mammalian cells; b) exposing the mammaliancells incubated with the immunogenic peptide to T cells; and c)screening for T cell recognition of the peptide using the mammaliancells incubated with the immunogenic peptide, whereupon theimmunogenicity of the peptide is determined.
 7. A method of detecting agp100 immunogenic peptide in a biological sample, wherein said peptideconsists of an amino acid sequence selected from SEQ ID NOs: 14-16 and20-21, comprising: a) contacting a reagent which specifically reacts andforms a complex with the gp100 peptide in said sample; and b) detectingthe formation of the complex between the peptide and the reagent,whereupon the gp100 immunogenic peptide in the biological sample isdetected.
 8. A method of preventing or inhibiting a recurrence ofmelanoma in a mammal comprising administering an effective amount of animmunogenic peptide consisting of a gp100 amino acid sequence selectedfrom SEQ ID NOs: 14-16 and 20-2, effective to stimulate animmunoprophylactic response to the melanoma, whereupon the recurrence ofthe melanoma is prevented or inhibited.
 9. The method of claim 7,wherein the sample is selected from the group consisting of mammaliantissues, mammalian cells, necropsy samples, pathology samples and biopsysamples.
 10. The method of claim 7, wherein the biological sample isfrom a mammal afflicted with a disease.
 11. The method of claim 10,wherein the biological sample is from a mammal afflicted with melanomaor metastatic melanoma.
 12. The method of claim 7, wherein the reagentis an antibody or fragment thereof.
 13. The method of claim 12, whereinthe reagent is a monoclonal antibody.
 14. The method of claim 12,wherein the reagent is a polyclonal antibody.
 15. The method of claim 7,wherein the detection of the complex is used to diagnose, assess orprognose a disease state.